1, 3, 17-trioxygenated androstane derivatives



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Patented May 6, 1958 1,3,17-TRIOXYGENATED AN DROSTANE DERIVATIVESRaymond M. Dodson, Park Ridge, Arthur H. Goldkamp, Deerfield, andRaphael Pappo, Skokie, 111., assignors to G. D. Searle & Co., Chicago,11]., a corporation of Delaware No Drawing. Application August 2, 1957Serial No. 675,808

7 Claims. (Cl. 26ll397.4)

The present invention relates to 1,3,17-trioxygenated androstanederivatives. The compounds of this invention can be represented by thegeneral structural formula CH3 CH i/Z 'clohexanecarbonyl,cyclohexaneacetyl, cyclopentanepropionyl, benzoyl, phenylacetyl,methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfonyl, andisomers and homologs of the foregoing. In the foregoing structuralformula the term Z can represent the carbonyl group (CO), thehydroxymethylene group (CHOH), a group of the formula CHO-Acyl whereinthe acyl radical can be selected from among the class of acyl radicalspreviously defined, or a group of the formula (lower)alkyl wherein thealkyl radical is a straightor branched-chain alkyl radical containingfewer than nine carbon atoms.

A suitable starting material for the manufacture of compounds of thisinvention is 1,3-dihydroxy-5-androsten-17-one(l-hydroxydehydroisoandrosterone) which can be obtained, as described incopending application Serial No. 624,958, filed November 29, 1956, bysubjecting dehydroisoandrosterone, under controlled conditions, to theoxygenating activity of a particularPenicillium organism isolated from asample of Illinois soil.

A culture of this microorganism has been deposited with the AmericanType Culture Collection, Washington, D. C., and has been designated asPenicilliurn sp., Accession No. 12,556. This organism is sometimesreferred to hereinafter as ATCC 12,556.

Other useful starting materials for the manufacture of compounds of thisinvention are esters of 1,3-dihydroxy- S-androsten-17-one, as well asvarious stereoisomeric 5- androstene-1,3,17-triols and esters thereof.

Hydrogenation of such a 1,3,l7-trioxygenated-5-androstene derivative inthe presence of a noble metal hydrogenation catalyst such as onecomprising platinum or palladium results in the saturation of thenuclear double bond and the formation of a corresponding androstanederivative.

Free hydroxyl groups present in the hydrogenated material can, ifdesired, be esterified by such means as treating the hydroxylic compoundin pyridine solution with a suitable acylating agent. Such suitableacylating agents can be selected from among the anhydrides and acidchlorides of hydrocarbon carboxylic acids and hydrocarbon sulfonicacids.

As differential rates of esterification are exhibited by hydroxyl groupsat positions 1, 3, and 17, there can conveniently be obtained eitherpartially esterified or completely esterified derivatives. For example,a 3 3- hydroxyl group is esterified at a substantially more rapid ratethan a let-hydroxyl group; and when a 1a,3 8-diol is subjected toreaction with acetic anhydride in pyridine solution at room temperature,the 3-monoacetate is present in good yield after reaction times of froml-4 hours, while the 1,3-diacetate is present in good yield afterreaction times of about 72 hours.

By oxidizing hydroxylic compounds of this invention to the correspondingketones, and subjecting such ketones to stereoselective reducingprocesses, the various stereochemical modifications of thel,3,l7trioxygenated derivatives of this invention can be obtained.Illustrative of this procedure is the reaction sequence, described morefully in the examples to follow, which comprises the oxidation ofla-hydroxy-3,B-acetoxy-5-androsten-l7-one to3fi-acetoxy-5-androstene-1,l7-dione,. reduction with lithium aluminumhydride to yield a reaction mixture from which5-androstene-l5,3fl,17B-triol is isolated, and catalytic hydrogenationto androstane-lfififi,l7,B-triol. In carrying out such a sequence, it issatisfactory that the double bond at position 5 be hydrogenated eitherprior to or subsequent to the chemical operations at position 1.

l7-alkyl derivatives encompassed within this invention can be preparedby the reaction of a l7-oxoandrostane derivative with an organometallicreagent. For example, reaction of 1a,3fi-dihydroxyandrostan-17-one withan alkylmagnesium halide, followed by hydrolysis of the primary reactionproduct, affords principally a 17-alkylandrostane-la,3,8,17B-triol. Thesame compound is obtained by catalytic hydrogenation of thecorresponding l7-alkyl-5-androstene-la,3fi,l7/3-triol.

The androstane derivatives of this invention have useful pharmacologicalproperties, as illustrated by their effects upon the central nervoussystem. Thus, they are barbiturate potentiators, as their administrationcauses a prolongation of the sleeping period induced by hexobarbital. Inaddition, they display toxicity toward certain lower forms of life, andcanjbe employed as anti-fungal agents. Thus, they are effective bytopical application in inhibiting the growth of T richophytonmentagrophytes.

The l7-oxoandrostane compounds afford pharmacologically-usefull7-ethynyl-17-hydroxy derivatives by reaction with acetylene in thepresence of a condensation catalyst such as potassium tertiary amylate.

This invention will appear more fully from the examples which follow.These examples are set forth by way of illustration only, and it will beunderstood that the invention is not to be construed as limited inspirit or in scope by the details contained therein, as manymodifications in materials and methods will be apparent from thisdisclosure to those skilled in the art. In these examples,

temperatures are given in degrees centigrade C.) and quantities ofmaterials, except as otherwise noted, in parts by weight.

Example 1 A stainless steel fermentation tank having a capacity of about40 liters is charged with a solution of 1000 grams of dextrose, 200grams of a cottonseed protein concentrate, 90 milliliters of corn steepliquor and 12 milliliters of concentrated hydrochloric acid in 25 litersof tap water. Five gramsof an anti-foaming agent, suitably of asilicone-type, is added and the contents of the vessel are sterilized bythe introduction of live steamunder pressure to a final temperature ofabout 110115 C. and'a final volume of about 30 liters. After cooling, aninoculation is made with a spore suspension of Penicillium sp.,'ATCC12,556, and the contents of the fermcntor are kept in agitation by astirrer operating at about 200 revolutions per minute. A stream of airwhich has been sterilized by filtration through a glass wool filter isintroduced through an inlet tube into the fermentor at a rate of about10 liters per minute. This rate of aeration is measured by means of arotameter placed in the sterile portion of the air line. Growth of theorganism is allowed to continue for 24 hours at about 25 C., duringwhich time additional small quantities of anti-foaming agent are addedif required. At this point about 20 liters of the culture is conductedby means of a sterile hose connection into a second stainless steelfermentation tank, which has a capacity of about 400 liters and whichhas previously been charged with about 255 liters of a sterilized andcooled aqueous solution of 1200 milliliters of corn steep liquor and 408grams of monobasic potassium phosphate, KH PO The contents of thisfermentor are kept agitated by a stirrer operating at about 150revolutions per minute and by the introduction of a stream of sterileair at a rate of about 30 liters per minute. Growthof the organism isallowed to proceed in this fermentor for an additional 24 hours, afterwhich time there is added a solution of 75 grams ofdehydroisoandrosterone in 1500 milliliters of acetone. Fermentation inthe presence of the'steroid substrate is continued for an additional 20hours at 25 C., with the same rates of stirring and aeration.Diatomaceous earth is then added, and the mycelium is separated bycentrifugation. The mycelium is stirred with 18 liters of methylenechloride, and the aqueous broth collected in the centrifugation of themycelium is'extracted with a total of 200 liters of methylene chloridein two equal portions. The combined methylene chloride solutions areconcentrated by distillation under reduced pressure to a suspensionhaving a small volume, and the suspension is then evaporated to dryness.The non-volatile residue is extracted with 200 milliliters of boilingether. Following this operation, which serves to remove certainether-soluble impurities, a solid or semisolid product amounting toabout 25 grams is collected on a filter. Upon crystallization of thiscrude product from a mixture of pyridine and ether and then from aqueouspyridine there is obtained purified 1m,3,B-dihydroxy-5-androsten-l7-one(1a-hydroxydehydroisoandrosterone) which melts at about 288-290 C. afterprior softening and hasa specific rotation 'of about +10.6 in chloroformsolution. The structural formula is The combined crystallization liquorsare concentrated to dryness, and a benzene solution of the non-volatileresidue is applied to a silica gel chromatography column containing aquantity of silica equal to 80 times the weight of the crystallizationliquor residues. By elution of the column with mixtures of benzene andethyl'acetate containing gradually increasing proportions of ethylacetate, there are obtained an additional quantity of :,35-

246.5 C. The specific rotation of this compound in ch10 4dihydroxy-S-androsten-l7-one, and a quantity oflot-hydroxy-4-androstene-3,17-dione. The latter compound is obtained ata satisfactory rate by elution with a 30-40 volume percent solution ofethyl acetate in benzene, and the former compound is obtained at asatisfactory rate by elution with a 50-70 volume percent solution ofethyl acetate in benzene.

Example 2 A suspension of 50 parts of l0:,3fi-dihYd1OXY-5-Bl1dl'05-ten-l7-one and 5 parts of 5% palladium on charcoal catalyst in 600 partsof ethanol is shaken in contact with a hydrogen atmosphere, withadditional portions of catalyst if required, for from 24 to 48 hours, oruntil a quantity of hydrogen at least approximately equal to onemolecular equivalent (in this example about 0.33 part) has beenabsorbed. ,The reaction mixture is filtered, and the filtrate is broughtto dryness by vaporization of the ethanol. Purification of the residueby recrystallization from a mixture of methanol and benzene afiords10:,35- dihydroxyandrostan-l7-one melting at about 202203.5 C. Thiscompound exhibits a specific rotation of about +88 in chloroformsolution; The structural formula is OH: on,

The same la,3pdihydroxyandrostan-l7-one is also obtained by thefermentation of 3fi-hydroxyandrostan-l7- one according to the method ofExample 1.

Example 3 A solution of 5 parts of 1a,3fl-dihydroxyandrostan-17- 7 on afilter and dried. Upon crystallization from a mixture of methylenechloride and ether the product obtained is1a,3fl-diacetoxyandrostan-17-one melting at about 244 roform solution isabout +77. The structural formula is A solution of 1 part of1a,3p-dihydroxy-5-androsten- 17-one in 20 parts of warm pyridine iscooled to room temperature and treated with 10 parts of aceticanhydride.

, The reaction mixture is allowed to stand at about 25 C.

for 1 hour and then filtered from a small quantity of insoluble residue.The filtrate is stirred with several times its volume of ice, until theice melts and sepa-' 6.7 in chloroform solution. This compound can alsobe purified by chromatography on a silica gel column, by elution withmixtures of benzene and ethyl acetate containing gradually increasingproportions of ethyl acetatc. The desired compound is eluted at asatisfactory rate with a 20 volume percent solution of ethyl acetate inbenzene. The structural formula is CHaCOO- Example 5 A solution of 1part of 141,3 ,B-dihydroxy-S-androsten-l7- one in 20 parts of warmpyridine is cooled to room temperature and treated with parts of aceticanhydride.

The reaction mixture is allowed to stand at about 25 C.

for 72 hours, after which it is stirred with several times its volume ofice until all of the ice has melted .and precipitation of the insolubleproduct is complete. This product is collected on a filter and thencrystallized a few times from methanol and from acetone. The purifiedcompound, 1a,3fl-diacetoxy-5-androsten-l7-one, melts at.

about 226227 C. and has a specific rotation of about +25 in chloroformsolution. This compound can also be purified by fractionation of thecrude product on a silica gel chromatography column, by eluting thecolumn with mixtures of benzene and ethyl acetate containing graduallyincreasing proportions of ethyl acetate. The desired compound is elutedfrom the column at a satisfactory rate with a 5 volume percent solutionof ethyl acetate in benzene. The structural formula is CH3 CH3 CHaC 0 QCHaCO 0 Example 6 Hydrogenation of 1a-hydroxy-35-acetoxy-5-androsten-17-one according to the procedure of Example 2 aifordslwhydroxy-Sfl-acetoxyandrostan-17-one. This compound exhibits infraredabsorption maxima at about 2.83, 5.75, and 8.08 microns, and has thestructural formula CHaCOO Example 7 The mixture is poured into severaltimes its vol- 6 ume of ice water, and'the insoluble product iscollected on a filter, dried, and crystallized twice from mixtures ofether and petroleum ether. The compound obtained is lot-hydroxy-3B(p-toluenesulfonoxy) and-rostan 17 one which melts with decomposition atabout 150 C. The structural formula is p t on, O

D-CHz- CsHrS 020 By the foregoing procedure, with the substitution of 4parts of 2,4-dimethylbenzenesulfonyl chloride for the p-toluenesulfonylchloride, the compound obtained is lahydroxy-3/3 (2,4-'dimethylbenzenesulfonoxy)androstan- 17-one.

Example 9.

Two parts of 1ct,3fi-dihydroxyandrostan-l7-one is dissolved in 45 partsof warm pyridine. The solution is cooled to room temperature and treatedwith 6 parts of methanesulfonyl chloride. The reaction mixture isallowed to stand at about 25 C. for 72 hours. .It is then poured intoseveral times its volume of ice water,

and the insoluble product is collected and washed with water. it isla,3fi-dimethanesulfonoxyandrostan-l'7-one, which exhibits prominentinfrared absorption maxima at about 5.76, 7.53, and 8.50 microns.

. mula is C H; c i

CHgSOz? onssom Example 10 To a mixture of 5 parts of1a,3B-dihydroxyandrostanl7-one in parts of ethanol is added a solutionof 2 parts of sodium borohydride in 20 parts of Water and 80 parts ofethanol. The reaction mixture is allowed to stand at room temperaturefor l and /2 hours, and is then heated under reflux for 5 minutes toinsure completion of the reaction. The mixture is poured into ice waterand acidified with dilute hydrochloric acid. It

-is then refrigerated to cause complete separation of product. Theinsoluble product is collected on a filter and washed with water. It isandrostane-1a,3;9,17/3-tri0l which melts at about 238239 C. and has aspecific rotation of about +20 in chloroform solution. The structuralformula is I w Example 11 To a solution of 3.35 parts of chromiumtrioxide in 20 parts of pyridine is added a solution of 3.2 parts ofla-hydroxy-3fi-acetoxy-S-androsten-17-one in 30 parts of pyridine. Thereaction mixture is stirred at about 35 The structural for asssgraa;

C. for 4 hours, after which it is: poured intoseveral.

CH: CH:

Example 12 Over a period of about 30 minutes a solution of 4 parts of3fl-acetoxy-5-androstene-l,17-dione in 570 parts of anhydrous ether isadded to a solution of 7 parts of lithium aluminum hydride in 1100 partsof refluxing anhydrous ether. Heating under reflux is continued for anadditional 2 hours, after which acetone and water are added to decomposethe unreacted lithium aluminum hydride. The reaction mixture is dilutedwith an equal volume of ether and washed twice with saturated ammoniumchloride solution and twice with water. The ethereal solution is madeanhydrous and brought to dryness by distillation. The residue consistsof a mixture of a lfi-hydroxy compound with the la-hydroxy epimer. Toobtain a more highly purified lfl-hydroxy compound, the residue iscrystallized from mixtures of methanol and benzene and then frommixtures of methanol and chloroform. This compound, S-androstene-1;3,38,17 8-triol, exhibits a variable melting point depending upon the rateof heating. On a melting point block preheated to about 270 C., it meltswith slight decomposition at about 274278 C.; Whereas it melts over abroader and lower rangewhen it is heated from room temperature. Thespecific rotation is about -39.8 in chloroform solution. The structuralformula is on. OH

Example 13 crystallization of the residue from a mixture of ether andmethanol,- there is. obtained androstanelfi,3fi,17fi-triol melting atabout 220-223 C. The structural formula is CHI Example 14 A reactionmixture prepared from 3 parts of androstanelm,3B,I7fi-tri0l, 50 parts ofpyridine and 12 parts of acetic anhydride is heated at about 90-100" C.for 4 hours and then maintained at about 25 C. for an additional-48hours. It is cautiously diluted with 10 parts of water, withexternalcooling, until the unreacted acetic anhydride is hydrolyzed.Dilution with water is then continued until separation of the insolublereaction PIfOd'.

net is complete. The precipitated product is collected and washed withwater. It is androstane-lu,3p,l7fi-triol triacetate. This compoundexhibits infrared absorption maxima at about 5.74 and 8.0 microns. Thestructural formula is 0 o 0 on;

By the foregoing procedure, with the substitution 0. 3 parts ofandrostane-1fl,3}3,l7fi-n'iol for the androstane- 1a,3,B,17fl-triol, andwith the substitution of 14 parts of propionic anhydride for the aceticanhydride, the compound obtained is androstane-113,3fi,l7fl-trioltripropionate.

Example 15 To a solution of 3 parts of androstane-lu,3fi,17fl-triol inparts of pyridine is added 10 parts of ethanesulfonyl chloride. Thereaction mixture is allowed to stand at about 25 C. for 5 days,following which it is poured into several times its volume of ice water.The insoluble product is collected and washed with water. It is1cz,3,B,17fi-triethanesulfonoxyandrostane. This compound exhibitsinfrared absorption maxima at about 7.52 and 8.50 microns. Thestructural formula is OH OBOIOH OH OHIQHIBOIQ CHICHIS 0:0

Example 16 By the procedure of Example 15, with the substitution of 6parts of benzoyl chloride for the ethanesulfonyl chloride, the compoundobtained is 1a,3p,17p-tribenzoyloxyandr'ostanea Example 17 To a solutionof 3 parts of androstane-la,3,8,17fi-triol in 50 parts of pyridine isadded 18 parts of S-cyclopentylpropionyl chloride. The exothermicreaction is moderated with an external cooling bath, and the mixture isthen maintained at about 25 C. for 7 days. It is poured into severaltimes its volume of ice water, and the insoluble product is washed bydecantation with water and dissolved in ethyl acetate. The ethyl acetatesolution is washed successively with diluted hydrochloric acid, withdiluted potassium carbonate solution, and then with several portions ofwater. By concentration of the organic solution to dryness, there isobtained a residue of 10:,313, l 7,8-tricyclopentylpropionoxyandrostane.

Example 18 To a refluxing mixture of 6.5 parts of 1a,33-dihydroxyandrostan-l7-one in 450 parts of tetrahydrofuran is added a 3molar solution of methylmagnesium bromide in butyl ether containing atotal of about 36 parts of methylmagnesium bromide. The addition iscarried out over a period of about 20 minutes, after which refluxing iscontinued for an additional 3 hours. Excess methylmagnesium bromide isdecomposed by the addition of water to the cooled reaction mixture. Themixture is then chilled and partitioned between ether and an excess ofsaturated ammonium chloride solution. The separated ethereal phase iswashed twice with saturated ammonium chloride solution and twice withwater, after which is is rendered anhydrous, filtered and concentratedby vaporization of the solvents under reduced pressure. The productwhich remains, can be crystallized from a mixture of ethyl acetate andpetroleum ether, and is 17- methylandrostane-lu,3,B,l7/3-triol which hasa prominent infrared absorption maximum at about 2.9 microns but lackssignificant infrared absorption in the wavelength range of 5.7-6.1mcirons. The structural formula is The same compound is obtained by thecatalytic hydrogenation of 17-methyl-5-androstene-lu,3B,17 3-trio1according to the procedure of Example 2.

When, in the foregoing procedure, an equivalent quantity ofethylmagnesium bromide is substituted for the methylmagnesium bromide,the compound obtained is 17-ethylandrostane-1a,3B,17;8-triol.

Example 19 To a refluxing solution of 1.25 parts of 1a,3/3-dihydroxy-5-androsten-17-one in 90 parts of tetrahydrofuran there is added asolution of 2 parts of lithium aluminum hydride in 45 parts oftetrahydrofuran over a period of about minutes. The reaction mixture isheated under reflux for an additional 20 minutes, after which it iscooled and treated in succession with 30 parts of ethyl acetate, 12parts of ethanol and 10 parts of water. The mixture is then diluted withether, and washed with two portions of 10% sodium hydroxide solution andwith water. The separated organic phase is dried over anhydrous sodiumsulfate, filtered, and concentrated to dryness by vaporization of thesolvents. The nonvolatile residue is crystallized from a mixture ofacetone and benzene to afiord purified S-androstene-1a,3fi,17}3-triolwhich, after thorough drying, melts at about 213-215 C. and has aspecific rotation of about 54.8 in chloroform solution. The

structural formula is CH3 OH By the hydrogenation of5-androsten-la,3;8,l713-triol according to the procedure of Example 2,the compound obtained is androstane-la,3 8,l7B-triol, identical with theproduct of Example 10.

What is claimed is:

l. A compound of the structural formula fin on wherein R and R aremembers of the class consisting of hydrogen and the acyl radicals ofhydrocarbon carboxylic acids and hydrocarbon sulfonic acids having fewerthan nine carbon atoms; and Z is a member of the class consisting of thecarbonyl group, the hydroxymethylene group, groups of the formulaCHO-acyl CH3 OH 4. Androstane-15,35,17B-trio1.

5. 1a,3fi-dihydroxyandrostan-17-one.

6. A compound of the structural formula wherein A and A are loweralkanoyl radicals.

7. 1u,3B-diacetoxyandrostan-17-one.

No references cited.

1. A COMPOUND OF THE STRUCTURAL FORMULA